Protein-protein interactions are an essential key in all biological processes, from the replication and expression of genes to the morphogenesis of organisms. Protein-protein interactions govern, among other things, ligand-receptor interactions and signaling pathways. The protein-protein interactions are also important in the assembly of enzyme subunits, in the formation of biological supramolecular structures, such as ribosomes, filaments and virus particles, and in antigen-antibody interactions.
Several approaches have been developed in attempts to identify protein-protein interactions. Some of the first techniques included co-purification of proteins and co-immunoprecipitation. However, these techniques are tedious and do not allow high throughput screening. Moreover, these techniques require lysis which corrupts the normal cellular context. A major breakthrough in the study of protein-protein interactions was achieved with the introduction of genetic approaches. Of the various genetic approaches, yeast two-hybrid (Fields and Song, 1989) is the most important one.
U.S. Pat. No. 5,637,463 describes an improvement of the yeast two-hybrid system which may be used to screen for modification-dependent protein-protein interactions. However, this method relies on the co-expression of a modifying enzyme which may exert its activity in the cytoplasm of the host organism. In this manner, the modifying enzyme may modify enzymes other than the protein involved in the protein-protein interaction and may affect the viability of the host organism.
Although the yeast two-hybrid system has been widely used, it has several drawbacks. One drawback is that the fusion proteins need to be translocated to the nucleus, which may not be evident. Also, proteins with intrinsic transcription activation properties may result in false positives. Moreover, protein-protein interactions that are dependent on secondary modifications of the protein, such as phosphorylation, may not be easily detected. To overcome some of these problems, alternative protein-protein interaction systems have been developed.
One of these alternative systems is an approach based on phage display that avoids the nuclear translocation drawback. PCT International Publication WO 9002809 describes an approach where a binding protein can be displayed on the surface of a genetic package, such as a filamentous phage, and the gene encoding the binding protein is packaged inside the phage. Phages bearing the binding protein that recognizes the target molecule are isolated and amplified. Several improvements of the phage display approach have been proposed and are as described in PCT International Publications WO 9220791, WO 9710330 and WO 9732017.
However, the phage display approaches suffer from difficulties inherent in phage display methodology. For instance, the proteins need to be exposed at the phage surface where the proteins are exposed to an environment that may not be physiologically relevant for the in vivo interaction. Moreover, when a phage library is screened, a competition exists between the phages which results in a selection of the high affinity-binding proteins. Also, modification-dependent phage display systems have not been described.
U.S. Pat. No. 5,776,689 describes a protein recruitment system which detects protein-protein interactions by recruitment of a guanine nucleotide exchange factor (Sos) to a plasma membrane where Sos activates a Ras reporter molecule. Activation of the Ras reporter molecule results in the survival of the cell that otherwise would not survive in the conditions used to culture the cell. Although this method allows the protein-protein interaction to take place under physiological conditions in the submembranary space, it has several drawbacks. For instance, modification-dependent interactions cannot be detected. Moreover, the method uses the pleiotropic Ras pathway and may cause technical complications.